A method of amplifying a target nucleic acid

ABSTRACT

The present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) multiple primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid and a kit used for the method. The present disclosure further provides a method of sequencing a target nucleic acid and a kit used for the method.

BACKGROUND OF THE INVENTION

Human genetic mutations whether it is de novo or somatic are critical information to understand human genetic disease (Ku, C. S. et al, A new era in the discovery of de novo mutations underlying human genetic disease, Hum Genomics 6, 27, 2012), cancer biology (Helleday, T. et al, Mechanisms underlying mutational signatures in human cancers, Nat Rev Genet 15, 585-598, 2014) and potential anticancer therapies. de novo mutation has long been known to cause genetic disease and it also plays an important role in rare and common forms of neurodevelopmental diseases, including intellectual disability, autism and schizophrenia (Veltman, J. A. et al, De novo mutations in human genetic disease, Nat Rev Genet 13, 565-575, 2012). Somatic mutation in cancer genome has been extensively studied and believed to hold the key to understand cancer origin, risk and potential biomarker discovery for therapeutic use. Detection of those genetic mutations is critical for diagnosis of disease and patient treatment.

Studies of de novo or somatic mutations in the human genome have been very challenging in the past because of genomic sequencing technology limitations. However, the development of high-throughput next-generation sequencing (NGS) technologies has greatly facilitated the study of such mutations. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can now be performed on parent offspring trios to identify de novo point mutations in the entire genome or within protein-coding regions, respectively.

WGS and WES are great tools for genetic mutation study, but they are still cost prohibitive for routine clinical use. In some cases, if only a set of genetic mutations are known to be related with certain disease or particular drug response, it would be efficient and cost effective to do genetic analysis for those genes. In order to do a limited resequencing of panel of genes, those genes need to be captured before carrying out NGS. The capture process could be achieved using either hybridization or amplicon approach. For hybridization capture approach, gDNA was first physically fragmented or enzymatically digested, then synthetic oligonucleotides are hybridized to regions of interest in solution to capture the intended sequences. For amplicon based approach, the intended regions are directly captured by amplification of PCR primers. Hybridization capture approach is scalable to large number of genes, but hybridization step usually takes overnight and the total process takes multiple days. It also requires at least 1 to 2 μg of gDNA material input. Amplicon based approach takes less time and only require 10 to 50 ng gDNA input, so it is suitable if quantities of DNA input from clinical samples are limited. However, multiplex PCR primers also generate nonspecific amplification products especially when the number of PCR primers increase. In fact, majority of PCR products are nonspecific amplicons when the number of primers approaches hundreds. Therefore, amplicon based approach usually uses an enzyme digestion step to reduce nonspecific amplification product followed by additional ligation step or use a multiple steps of cleaning up to reduce those nonspecific products. Those nonspecific amplification products not only require multiple steps during sequencing library generation but also can introduce sequencing data errors.

Recently detection of low frequency mutation has been a rapidly growing area of interest because of its important applications in basic and clinical research. One kind of rare mutations, circulating cell-free DNA (cfDNA) from human plasma are used for prenatal screening (Chiu, R. W. et al, Noninvasive prenatal diagnosis empowered by high-throughput sequencing, Prenat Diagn 32, 401-406, 2012), while circulating tumor DNA (ctDNA) has been confirmed to contain the hallmark mutations of cancerous cells. ctDNA has the potential to be a novel, non-invasive biomarker that promotes early cancer detection at a surgically curable stage, reduces the necessity of repeat tissue biopsies, and detects the early relapse of the disease, thereby increasing the efficacy of targeted therapy. For cancers that are often detected at a late stage, including lung, pancreatic, and ovarian etc., a high-sensitivity ctDNA assay could be used as an important screening test to detect typically terminal metastatic stage cancer at an earlier, potentially curable stage. With continuous ctDNA monitoring from patient blood, change of ctDNA composition and quantitation could be used to monitor cancer progression in real time, improving patient safety and eliminating the cost related to repeat tissue biopsies.

Unfortunately, detection of ctDNA remains challenging by its presence in relatively low quantities especially in early-stage cancer patients. There are several available techniques developed so far to detect ctDNA including BEAMing, digital PCR, and next generation sequencing. All those methods can detect low frequency mutations by assessing individual molecules one-by-one. NGS has the advantage over traditional methods in that large amount of sequencing information can be obtained easily in an automated fashion. However, NGS cannot generally be used to detect rare mutations because of its high error rate associated with NGS library generation and the sequencing process. Some of these errors presumably result from mutations introduced during template preparation, during the pre-amplification steps required for library preparation and during further solid-phase amplification on the instrument itself. Other errors are due to base mis-incorporation during sequencing and base-calling errors.

Therefore, there remains a continuing need for a novel approach to eliminate nonspecific amplification products during multiplex PCR reaction so that the sequencing library could be directly generated without additional digestion and. ligation steps, and a novel approach to reduce error rate so that rare mutation could be reliably detected using current NGS instrument.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleicacid polymerase using the blocking primers; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid,

In some embodiments, the blocking group is at or near 3′ terminal of each blocking primer. In some embodiments, the blocking group is 2′, 3′-dideoxynucleotide, ribonucleotide residue, 2′, 3′ SH nucleotide, or 2′-O—PO₃ nucleotide.

In some embodiments, the blocking primer is complementary to a portion of the target nucleic acid. In some embodiments, the blocking primer is further modified to decrease the amplification of undesired nucleic acid. In some embodiments, the modification is introduction of at least one mismatched nucleotide in the primer. In some embodiments, the mismatched nucleotide is 2-18 bp away from the nucleotide with the blocking group. In some embodiments, wherein the mismatched nucleotide is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 bp away from the nucleotide with the blocking group. In some embodiments, the mismatched nucleotide base is located on the 5′ side of the nucleotide with the blocking group, in some embodiments, the modification is a modification to decrease the Tm between the blocking primer and the undesired nucleic acid. In some embodiments, the modification is a modification to increase the Tm between the blocking primer and the target nucleic acid. In some embodiments, wherein the modification is a modification to form an extra bridge connecting the 2′ oxygen and 4′ carbon of at least one nucleotide of the blocking primer.

In some embodiments, there are no more than 20 complementary nucleotide pairings and no more than 50% sequence complementarity between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 complementary nucleotide pairings between any two primers.

In some embodiments, the reaction mixture comprises at least 30, 40, 50, 60, 70, 80 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 different types of primer pairs.

In some embodiments, each of the primers is 8 to 100 nucleotides in length.

In some embodiments, the different types of primer pairs can complementarily bind to different target nucleic acids or different sequences in the same target nucleic acid.

In some embodiments, wherein the de-blocking agent is CS5 DNA polymerase with the mutations selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G or the combination of such mutations, ampliTaq or KlenTaq polymerase with F667Y mutation, pyrophosphate or RNase H2.

In some embodiments, the target nucleic acid is single stranded or double stranded DNA.

In some embodiments, the target nucleic acid is double stranded DNA ligated with single or double adaptor tags or single stranded DNA ligated with single adaptor tag.

In some embodiments, the reaction mixture further comprises at least one primer complementary in whole or in part with the adaptor tag.

In some embodiments, the target nucleic acid is double stranded DNA comprising single or double molecular index tag or single stranded DNA comprising single molecular index tag. In some embodiments, the molecular index tag comprises unique identifier nucleic acid sequence and an adaptor tag.

In some embodiments, the primers have common tailing sequence at or near 5′ terminal of the primers. In some embodiments, the common tailing sequence can be used as molecular index tag, sample index tag or adaptor tag or combinations of all three tags.

In some embodiments, the reaction mixture further comprises high fidelity polymerase. In some embodiments, the high fidelity polymerase is PFU DNA Polymerase.

In some embodiments, the step (b) “incubating the reaction mixture under a condition for amplification of the target nucleic acid” comprises the steps of denaturing the target nucleic acid; annealing the primers with the target nucleic acid to allow the formation of a nucleic acid-primer hybrid; and incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid.

In some embodiments, the formation of a nucleic acid-primer hybrid results in de-blocking the block group in the primer through de-blocking agent.

In some embodiments, the steps of “denaturing the target nucleic acid; annealing the primers with the target nucleic acid to allow the formation of a nucleic acid-primer hybrid; and incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid” is repeated at least 1 time, 5 times; 10 times, 15 times, 20 times, 2.5 times, 30 times, 35 times, 40 times or 50 times. In some embodiments, the step (b) is repeated from about 20 times to about 50 times.

In some embodiments, the nucleic acid sample comprises the target nucleic acid. In some embodiments, the target nucleic acids in the nucleic acid sample is no more than 1 copy, 2 copies, 5 copies, 8 copies, 10 copies, 20 copies, 30 copies, 50 copies, 80 copies or 100 copies. In some embodiments, the molar percentage of target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%.

In some embodiments, the nucleic acid other than the target nucleic acid is not amplified in step (b) substantially. In some embodiments, the molar percentage of undesired nucleic acid in the reaction products obtained from step (b) is less than 20©0 15%, 10%, 5%, 3%, 2% or 1%.

In some embodiments, the method is used for selective enrichment of mutant nucleic acid in a sample comprising wildtype nucleic acid. In some embodiments, wherein at least one blocking primer is complementary to the mutant nucleic acid at the mutant residues and the nucleotide of the blocking primer corresponding to a mutant residue has the blocking group.

Another aspect of the present disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; and (c) determining the sequence of the reaction products obtained from step (b).

In some embodiments, the method is used for sequencing by capillary electrophoresis, PCR or high throughput sequencing, In some embodiments, the blocking primer is further modified to decrease the amplification of undesired nucleic acid.

In some embodiments, the reaction mixture further comprises high fidelity polymerase.

Yet another aspect of the present disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; (c) adding adaptor tag, molecular index tag and/or sample index tag to the reaction products obtained from step (b); and (d) determining the sequence of the reaction products obtained from step (c).

In some embodiments, the method is used for sequencing by capillary electrophoresis, PCR or high throughput sequencing.

In some embodiments, wherein the blocking primer is modified to decrease the amplification of undesired nucleic acid.

In some embodiments, wherein the reaction mixture further comprises high fidelity polymerase.

Yet another aspect of the present disclosure provides a kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (ii) nucleic acid polymerase, and (iii) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primer.

In some embodiments, the blocking primer is modified to decrease the amplification of undesired nucleic acid.

In some embodiments, the reaction mixture further comprises high fidelity polymerase.

Yet another aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least one type of primers that is complementary to a portion of the target nucleic acid, and each type of primers has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to decrease the amplification of undesired nucleic acid, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primer; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid.

In some embodiments, the modification is introduction of at least one mismatched nucleotide in the primer.

In some embodiments, the mismatched nucleotide is 2-18 bp away from the nucleotide with the blocking group.

In some embodiments, the mismatched nucleotide is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 by away from the nucleotide with blocking group.

In some embodiments, the mismatched nucleotide is located on the 5′ side of the blocking group.

In some embodiments, the modification is a modification to decrease the affinity between the blocking primer and the target nucleic acid.

In some embodiments, the modification is a modification to form an extra bridge connecting the 2′ oxygen and 4′ carbon of at least one nucleotide of the blocking primer.

In some embodiments, the method is used for selective enrichment of mutant nucleic acid in a sample comprising wild type nucleic acid.

In some embodiments, a blocking primer is complementary to a portion of the target nucleic acid. In some embodiments, the blocking primer is complementary to the mutant nucleic acid at the mutant residue and the nucleotide of the blocking primer corresponding to a mutant residue has the blocking group.

Yet another aspect of the present disclosure provides a kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least one type of primers that is complementary to a portion of the target nucleic acid, and each type of primers have at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to decrease the amplification of undesired nucleic acid, (ii) nucleic acid polymerase, and (iii) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primer.

In some embodiments, the blocking primer is modified to decrease the affinity between the blocking primer and the target nucleic acid.

BRIEF DESCRIPTION OF THE :DRAWINGS

FIG. 1: NGS library construction for genomic DNA by multiplex PCR.

FIG. 2: NGS library construction for fragmented DNA by multiplex PCR.

FIG. 3: NGS library construction for fragmented DNA with single stranded molecular index tag by multiplex PCR.

FIG. 4: NGS library construction for fragmented DNA with double stranded molecular index tags by multiplex PCR.

FIG. 5: Selectively amplification of mutant sequence in genomic)NA by multiplex PCR.

FIG. 6: Mutant enriched NGS library construction for fragmented DNA by multiplex PCR.

FIG. 7: Mutant enriched NGS library construction for fragmented DNA with single stranded molecular index tag by multiplex PCR.

FIG. 8: NGS library construction for fragmented DNA with double stranded molecular index tags by multiplex PCR.

FIG. 9. The normalized reads per amplicon in a 196-plea reaction on a genomic DNA sample across six individual reactions followed by sequencing run on a MiSeq sequencer in Example 1.

FIG. 10. The normalized reads per amplicon v.s. amplicon GC percentage in a 196-plex reaction on a genomic DNA sample across six individual reactions followed by sequencing run on a MiSeq sequencer in Example 1.

FIG. 11. General working flow for multiplex PCR reaction assay design and NGS data analysis.

FIG. 12. Electropherogram of selectively enriched different mutant nucleic acids after multiplex PCR reaction in Example 2.

FIG. 13. Electropherogram of selectively enriched mutant nucleic acid after multiplex PCR reaction in Example 3.

FIG. 14. Electropherogram of selectively enriched mutant nucleic acid after multiplex PCR reaction in Example 4.

FIG. 15. The sketch of multiplex PCR and the construction of library in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid.

Providing a Reaction Mixture

In some embodiments, a reaction mixture for detecting a target nucleic acid of the present disclosure comprises: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers.

Nucleic Acid Sample

The term “nucleic acid” as used in the present disclosure refers to a biological polymer of nucleotide bases, and may include but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), micro RNA (miRNA), and peptide nucleic acid (PNA), modified oligonucleotides (e.g., oligonucleotides comprising nucleotides that are not conventional to biological RNA or DNA, such as 2′-O-methylated oligonucleotides), and the like. The nucleotides of the present disclosure can be natural or unnatural, substituted or unsubstituted, modified or unmodified. The nucleotides can be linked by phosphodiester bonds, or by phosphorothioate linkages, methyiphosphonate linkages, boranophosphate linkages, or the like. The polynucleotides can additionally comprise non-nucleotide elements such as labels, quenchers, blocking groups, or the like. The nucleic acid can be, e.g., single-stranded or double-stranded.

The term “DNA” as used in the present disclosure refers to deoxyribonucleic acid, a long chain polymer biological macromolecule which forms genetic instructions. The subunit of DNA is nucleotide. Each nucleotide in DNA consists of a nitrogenous base, a five-carbon sugar (2-deoxyribose) and phosphate groups. Neighboring nucleotides are linked via diester bonds formed by deoxyribose and phosphoric acid, thereby forming a long chain framework. Generally, there are four types of nitrogenous bases in DNA nucleotides, namely adenine (A), guanine (G), and cytosine (C), thyrnine (T). The bases on the two DNA long chains pair via hydrogen bonds, wherein adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C).

The term “nucleic acid sample” as used in the present disclosure refers to any sample containing nucleic acid, including but not limited to cells, tissues, and body fluids, etc. In some embodiments, the nucleic acid sample is a tissue, e.g., biopsy tissue or paraffin embedded tissue. In some embodiments, the nucleic acid sample is bacteria or animal or plant cells. In some other embodiments, the nucleic acid sample is a body fluid, e.g., blood, plasma, serum, saliva, amniocentesis fluid, pleural effusion, seroperitoneum, etc. In some embodiments, the nucleic acid sample is blood, serum or plasma.

In some embodiments, the nucleic acid sample comprises or is suspected of comprising the target nucleic acid.

The term “target nucleic acid” or “target region” as used in the present disclosure refers to any region or sequence of a nucleic acid which is to be amplified intentionally.

In some specific embodiments, the target nucleic acid is DNA, RNA or a hybrid or a mixture thereof. In some specific embodiments, the target nucleic acid is genomic DNA. In some specific embodiments, the target nucleic acid is cell-free DNA (cfDNA). In some specific embodiments, the target nucleic acid is circulating tumor DNA (ctDNA).

“Cell-free DNA” as used in the present disclosure refers to DNA released from cells and found in circulatory system (e.g., blood), the source of which is generally believed to be genomic DNA released during apoptosis.

“Circulating tumor DNA” as used in the present disclosure refers to the cell-free DNA originated from tumor cells. In human body, a tumor cell may release its genomic DNA into the blood due to causes such as apoptosis and immune responses. Since a normal cell may also release its genomic DNA into the blood, circulating tumor DNA usually constitutes only a very small part of cell-free DNA.

In some embodiments, the target nucleic acid is single stranded or double stranded DNA. In some embodiments, the target nucleic acid is the whole or a portion of one or more genes selected from ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53 and VHL.

In some embodiments, the amount of target nucleic acid in the nucleic acid sample is no more than 1 copy, 2 copies, 3 copy, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, 10 copies, 12 copies, 15 copies, 18 copies, 20 copies, 30 copies, 50 copies, 80 copies or 100 copies. In some embodiments, the molar percentage (molar/molar) of target nucleic acid in the nucleic acid sample is less than 50%, 20%, 105, 8%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005% or 0.001%. In some embodiments, the ratio of molar of target nucleic acid and the molar of un-target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 8%, 6%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%.

In some embodiments, the target nucleic acid is DNA fragment. In some embodiments, the size of the target nucleic acid is 0.01-5 kb, 0.1-5 kb, 0.1-1 kb, 1-2 kb, 2-3 kb, 3-4 kb, 4-5 kb, 0.2-0.4 kb, 0.5-1 kb, 0.1-0.5 kb, 0.01-0.5 kb, 0.01-0.4 kb, 0.01-0.3 kb, 0.01-0.25 kb, 0.02-0.25 kb, 0.05-0.3 kb or 0.05-0.25 kb. The DNA fragment can be obtained through common technology in the art (e.g., physical breaking, cleavage using specific restriction endonuclease, etc.).

In some embodiments, the target nucleic acid is double stranded DNA ligated with single or double adaptor tags or single stranded DNA ligated with single adaptor tag.

The term “adaptor tag” as used in the present disclosure refers to a specific DNA sequence attached to one or two ends of a nucleic acid (single stranded or double stranded) according to needs, and the length of the adaptor is usually within 5-50 bp. The adaptor tag can be used to facilitate amplifying the target nucleic acid and/or sequencing the amplified target nucleic acid. In some embodiments, the adaptor tag is used to facilitate the ligation of tags for sequencing (e.g., the ligation of P5 and P7 tag for Illumina MiSeq sequencer). In some embodiments, the adaptor tag is attached to only one end of a single stranded nucleic acid at 3′ terminal or 5′ terminal. In some embodiments, the adaptor tag is attached to two ends of a single stranded nucleic acid. In some embodiments, one adaptor tag is attached to each strand in double stranded nucleic acid at 3′ terminal or 5′ terminal. For example, one adaptor tag is attached to one strand in double stranded nucleic acid at its 3′ terminal and one adaptor tag is attached to the other strand in double stranded nucleic acid at its 5′ terminal, and the two adaptor tags are identical or complementary to each other. In some embodiments, two adaptor tags are attached to two ends of each strand in double stranded nucleic acid.

The adaptor tag can be attached to the nucleic acid through common technologies in the art. in some embodiments, where the target nucleic acid is double stranded DNA, the adaptor tag can he attached to the nucleic acid through the following steps: (a) providing an adaptor ligation nucleic acid designed to contain sequences to ligate with an end of one strand of the DNA (for example, the adaptor ligation nucleic acid contains a hybridization complementary region, or a random hybridization short sequence, e.g., poly-T); (b) hybridization of the adaptor ligation nucleic acid and the strand of the DNA; and (c) adding polymerase (e.g., reverse transcriptase) after the hybridization to extend the adaptor ligation nucleic acid, thereby the adaptor tag is ligated to the end of the target DNA fragment. For attaching another adaptor to the other end of the same strand or to the other strand of the DNA, an adaptor ligation nucleic acid can be designed according to the needs and steps (b)-(c) can be repeated. In some other embodiments, where the DNA fragment is double stranded and the end of the DNA fragment is a sticky end, the adaptor tag can be attached to the nucleic acid through the following steps: (a) designing the adaptor ligation nucleic acid to contain sequences to ligate with the sticky end; (b) complementarily annealing the adaptor ligation nucleic acid with the sticky end; and (c) ligating the adaptor ligation nucleic acid to the double stranded of the target DNA using a ligase, thereby achieving the purpose of attaching the adapter to the end of the DNA fragment.

In some embodiments, the target nucleic acid is double stranded DNA comprising single or double molecular index tags or single stranded DNA comprising single molecular index tag. In some embodiments, the molecular index tag comprises unique identifier nucleic acid sequence and an adaptor tag. In some embodiments, the adaptor tag is at one end of the target nucleic acid.

The term“molecular index tag” as used in the present disclosure refers to a nucleic acid sequence used as a tag, which can be ligated to or existing at the 5′ end, the 3′ end or both ends of a DNA fragment. In DNA sequencing, especially in high throughout sequencing technology, a molecular index tag therein is used to mark particular DNA molecule. After amplification and sequencing, the count of the molecular index sequence therein is used to mark particular DNA molecule and can be the basis for determining the quantity of expression of the marked gene, or be used to trace the information of the amplified DNA molecules from the same original molecules and thereby correcting the random errors of DNA sequences during amplification and sequencing.

In some embodiments, the molecular index tag is exogenous, which is attached to the target nucleic acid through PCR (e.g., as described in MoCloskey M. L. et al, Encoding PCR products with batch-stamps and barcodes. Biochem Genet 45:761-767, 2014 or Parameswaran P, et al., A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Res 35:e130, 2017) or ligation (e.g., as described in Craig D W, et al., Identification of genetic variants using bar-coded multiplexed sequencing. Nat Methods 5:887-893, 2008 or Miner B E, et al, Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR. Nucleic Acids Res 32:e135, 2004). In some embodiments, the molecular index tag or the unique identifier nucleic acid sequence therein can be a random sequence (i.e., formed with randomly arranged A/T/C/G).

In some embodiments, the molecular index tag or the unique identifier nucleic acid sequence therein is endogenous, which are the sequences of the two ends of randomly sheared fragment.

More information for molecular index tag can be found in U.S. 20140227705 and U.S. 20150044687.

Primer

The term “primer” as used in the present disclosure refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. Primer may comprise natural ribonucleic acid, deoxyribonucleic acid, or other forms of natural nucleic acid. Primer may also comprise un-natural nucleic acid (e.g., LNA, ZNA etc.).

Primers may be prepared using any suitable method, such as, for example, the phosphotriester and phosphodiester methods or automated embodiments thereof In one such automated embodiment, diethylophosphoramidites are used as starting materials and may be synthesized as described by Beaucage et al., Tetrahedron Letters, 22:1859-1862 (1981). One method for synthesizing primer on a modified solid support is described in U.S. Pat. No. 4,458,006. It is also possible to use a primer which has been isolated from a biological source, such as a restriction endonuclease digest. In some embodiments, the primer with blocking nucleotide at the 3′ end, can be synthesized with terminal transferase (Gibco BRL) (Nuc Aci Res 2002, 30(2)).

The term “primer pair” as used in the present disclosure refers to a pair of primers consisting of a forward primer and a reverse primer which complement with a portion of a sequence to be amplified, respectively, wherein the forward primer defines a point of initiation of the amplified sequence and the reverse primer defines a point of termination of the amplified sequence. The term “complimentary”, when it is used to describe the relationship between primer and the sequence to be amplified, refers to that the primer is complimentary to the sequence to be amplified or is complimentary to a complementary sequence of the sequence to be amplified.

The pair of primers can be designed based on the sequence of the target nucleic acid. In some embodiments, at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid. in some embodiments, when the target sequence (assuming it is a double stranded DNA) has an adaptor tag, one primer of a primer pair may be complementary to a portion of the target sequence (on one strand) and the other primer may be complementary to the adaptor tag (on the other strand).

In some embodiments, each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension. In some embodiments, both primers in each primer pair are blocking primers comprising a blocking group capable of blocking polymerase extension.

The term “blocking primer” as used in the present disclosure refers to a primer having a blocking group.

The term “blocking group” as used in the present disclosure refers to any chemical group covalently linked in a nucleic acid chain and capable of blocking polymerase extension. in some embodiments, the nucleotide with blocking group is a modified nucleotide at or near the 3′ terminal of each blocking primer. In some embodiments, the nucleotide with blocking group is no more than 6 bp, 5 bp, 4 bp, 3 bp, 2 bp or 1 bp away from the 3′ terminal of each blocking primer. In some embodiments, when the method of the present disclosure is used for selective enrichment of mutant nucleic acid in a sample comprising wildtype nucleic acid, the blocking group is at the nucleotide that is complementary with the corresponding mutated nucleotide of the mutant nucleic acid but is not complementary with the corresponding nucleotide of wildtype nucleic acid.

In some embodiments, the blocking group is 2′, 3′-dideoxynucleotide, ribonucleotide residue, 2′, 3′SH nucleotide, or 2′-O—PO₃ nucleotide. When the blocking group is a ribonucleotide residue, the blocking primer is a primer that has one ribonucleotide residue and other residues are all deoxyribonucleotide residues.

More information for blocking group and blocking primer can be found in U.S. Pat. Nos. 9,133,491, 6,534,269 and Joseph R.D. et al., RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers, BMC Biotechnology 11:80, 2011.

In some embodiments, the blocking primer is complementary to a portion of the target nucleic acid.

In some embodiments, the primers are 5 to 100 nucleotides in length. In some embodiments, the primers are at least 5, 6, 7, 9, 10, 15, 20, 25, 30, 35 or 40 nucleotides in length. In some embodiments, the primers are no more than 100, 90, 80, 70, 60, 50, 40, 35, 30, 25 or 20 nucleotides in length.

In some embodiments, a primer comprises a complementary region that is complementary to the target sequence and a common tailing sequence at or near the 5′ terminal of the primer. In some embodiments, the common tailing sequence can be used as molecular index tag, adaptor tag or sample index tag or combinations of all the three tags.

The term “sample index tag” as used in the present disclosure refers to a series of unique nucleotides (i.e., each sample index tag is unique), and can be used to allow for multiplexing of samples such that each sample can be identified based on its sample index tag. In some embodiments, there is a unique sample index tag for each sample in a set of samples, and the samples are pooled during sequencing. For example, if twelve samples are pooled into a single sequencing reaction, there are at least twelve unique sample index tags such that each sample is labeled uniquely.

In some embodiments, the blocking primer is modified so as to further decrease the amplification of undesired nucleic acid.

In some embodiments, the modification is introduction of at least one mismatched nucleotide in the primer, In some embodiments, the mismatched nucleotide base is located on the 5′ side of the nucleotide with the blocking group.

The term “mismatched nucleotide” as used in the present disclosure refers to a nucleotide of a first nucleic acid (e.g., primer) that is not capable of pairing with a nucleotide at a corresponding position of a second nucleic acid (e.g., target nucleic acid), when the first and second nucleic acids are aligned.

The preferred or accepted location of the mismatched nucleotide can be determined through conventional technologies. For example, the mismatched nucleotides are introduced into different locations in the blocking primer, and those blocking primers are used for amplifying a target nucleic acid separately, and then the preferred or accepted location of the mismatched nucleotide for the target nucleic acid can be determined based on the results of amplification (e.g., the location decreasing the amplification of undesired nucleic acid or false positive results is preferred or accepted location). The location of the mismatched nucleotide may change along with the change of the target nucleic acid or the structure of the blocking primer. In some embodiments, the mismatched nucleotide is 2-18 bp away from the nucleotide with blocking group. in some embodiments, the mismatched nucleotide is 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 bp away from the nucleotide with blocking group. In some embodiments, the mismatched nucleotide is no less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 by away from the nucleotide with blocking group. In some embodiments, the mismatched nucleotide is no more than 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 bp away from the nucleotide with blocking group.

In some embodiments, the modification is a modification to increase the melting temperature (Tm) between the blocking primer and the target nucleic. In some embodiments, the modification is a modification to decrease the melting temperature (Tm) between the blocking primer and the undesired nucleic acid which may be the wildtype nucleic acid in a method for selective enrichment of mutant nucleic acid in a sample. In some embodiments, wherein the modification is a modification to form an extra bridge connecting the 2′ oxygen and 4′ carbon of at least one nucleotide of the blocking primer, such as locked nucleic acid (LNA), see, e.g., Karkare S et al., Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino. Appl Microbiol Biotechnol 71(5):575-586. 2006 and Vester B et al., LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43(12):13233-13241, 2004.

In some embodiments, the reaction mixture comprises at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 different types of primer pairs. In some embodiments, the different types of pairs of the primers are complementary to different target nucleic acid fragments or are complementary to different sequences in the same target nucleic acid fragment.

The inventors of the present disclosure conducted a simulation experiment to evaluate the probability of forming primer pairs between primers with certain lengths generated randomly. The inventors randomly generated from 10 to 490 primer pairs in length of 20 bp to form different primer pools and, for each pool, checked primer dimer formation between any one primer and other primer in the same pool. It can be seen that the probability to form primer dimer (e.g., resulting from complementarily between different primers) is increased along with the increasing numbers of primers.

TABLE 1 Relationship between the number of the primers and the dimer length. Number of Dimer Length Primer Pairs 4 bp 5 bp 6 bp 7 bp 8 bp 10 100%  90%  20%  0%  0% 30 100% 100%  67%  7%  7% 50 100% 100%  88% 22%  0% 70 100% 100%  94% 50% 20% 90 100% 100%  96% 48%  9% 110 100% 100% 100% 72% 13% 130 100% 100%  99% 61% 23% 150 100% 100%  99% 72% 23% 170 100% 100% 100% 79% 29% 190 100% 100%  99% 77% 32% 210 100% 100% 100% 82% 31% 230 100% 100% 100% 83% 34% 250 100% 100% 100% 87% 34% 270 100% 100% 100% 91% 35% 290 100% 100% 100% 90% 39% 310 100% 100% 100% 96% 49% 330 100% 100% 100% 96% 47% 350 100% 100% 100% 96% 51% 370 100% 100% 100% 95% 48% 390 100% 100% 100% 96% 53% 410 100% 100% 100% 97% 52% 430 100% 100% 100% 97% 54% 450 100% 100% 100% 97% 54% 470 100% 100% 100% 98% 59% 490 100% 100% 100% 98% 63%

For the data in Table 1, 100% means that each primer in a primer pool forms a dimer with at leak one of the other primers in the same primer pool and the length of the dimer is no shorter than the indicated number; 20% means that 20% of the primers in a primer pool forms dimers in the primer pool and the length of the dimer is no shorter than the indicated number.

TABLE 2 Relationship between the number of the primers and the dimer length in the 3' terminal of the primer Numbers of Dimer Length Primer Pairs 4 bp 5 bp 6 bp 7 bp 8 bp 10  50%  10%  0%  0%  0% 30  90%  50% 13%  7%  7% 50  98%  56% 26%  2%  0% 70  97%  67% 23%  7%  0% 90 100%  80% 33% 12%  6% 110 100%  88% 50%  7%  3% 130 100%  82% 40% 10%  4% 150 100%  91% 41% 13%  2% 170 100%  95% 49% 15%  4% 190 100%  94% 54% 15%  3% 210 100%  94% 50% 14%  3% 230 100%  97% 59% 20%  6% 250 100%  99% 58% 21%  7% 270 100% 100% 69% 23%  6% 290 100%  99% 65% 19%  5% 310 100%  99% 67% 22%  6% 330 100%  99% 70% 24%  4% 350 100% 100% 71% 25%  7% 370 100% 100% 73% 26%  6% 390 100% 100% 73% 27%  7% 410 100% 100% 75% 31% 10% 430 100% 100% 77% 36%  8% 450 100% 100% 79% 34%  7% 470 100% 100% 81% 31%  9% 490 100% 100% 82% 36%  9%

For the data in Table 2, 100% means that each primer in a primer pool forms a dimer from its 3′ terminal with at least one of other primers in the same primer pool and the length of the dimer is no shorter than the indicated number; 10% means that 10% of the primers in a primer pool forms dimers in the primer pool and the length of the dimer is no shorter than the indicated number.

In some embodiments, there are no more than 20 complementary nucleotide pairings between any two primers, In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 complementary nucleotide pairing between any two primers. In some embodiments, there are no more than 20 consecutive complementary nucleotide pairings between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 consecutive complementary nucleotide pairings between any two primers. In some embodiments, the above mentioned complementary nucleotides pairings are within a region from the 1^(st) nucleotide at the 3′ terminal of a primer to the 20^(th), 19^(th), 18^(th), 17^(th), 16^(th), 15^(th), 14^(th), 13^(th), 12^(th), 11^(th), 10^(th), 9^(th) or 8^(th) nucleotide from the 3′ terminal of the primer. In some embodiments, there are no more than 7, 6 or 5 consecutive complementary nucleotide pairings within a region from the 1^(st) nucleotide at the 3′ terminal of a primer to the 20^(th), 19^(th), 18^(th), 17^(th), 16^(th), 15^(th), 14^(th), 13^(th), 12^(th), 11^(th), 10^(th), 9^(th)or 8^(th) nucleotide from the 3′ terminal of the primer. In some embodiments, when calculating the number of parings between two primers, the common tailing sequence is not counted.

In some embodiments, there are no more than 20 complementary nucleotide pairings and no more than 50% sequence complementarity between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 complementary nucleotide pairings and no more than 45%, 40%, 35%, 30%, 25% or 20% sequence complementarity between any two primers. In some embodiments, when calculating the percent complementarity between two primers, the common tailing sequence is not counted.

The term “nucleotide complementarity” or “complementarity” when in reference to nucleotide as used in the present disclosure refers to a nucleotide on a nucleic acid chain is capable of base pairing with another nucleotide on another nucleic acid chain. For example, in DNA, adenine (A) is complementary to thymine (T), and guanine (G) is complementary to cytosine (C). For another example, in RNA, adenine (A) is complementary to uracil (U), and guanine (G) is complementary to cytosine (C).

The term “percent complementarity” as used in the present disclosure refers to the percentage of nucleotide residues in a nucleic acid molecule that have complementarity with nucleotide residues of another nucleic acid molecule when the two nucleic acid molecules are annealed. Percent complementarity is calculated by dividing the number of nucleotides of the first nucleic acid that are complementary to nucleotides at corresponding positions in the second nucleic acid by the total length of the first nucleic acid.

Percent complementarity of a nucleic acid or the number of nucleotides of a nucleic acid that is complementary to another nucleic acid can also be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 215, 403-410, 1990; Zhang and Madden, Genome Res., 7, 649-656, 1997).

For example, primer 1 in which 18 of 20 nucleotides of the primer 1 have complementarity with 18 nucleotides of primer 2 would have 90% sequence complementarity. In this example, the complementary nucleotides may be contiguous to each other or interspersed with non-complementary nucleotides.

The term “x nucleotide pairings” as used in the present disclosure refers to the number of nucleotide residues in a nucleic acid molecule that has complementarity with the corresponding nucleotides of another nucleic acid molecule when the two nucleic acid molecules are annealed. For example, “18 nucleotide pairings” means 18 nucleotide residues of a first nucleic acid molecule has complementarity with 18 nucleotide residues of a second nucleic acid molecule. In this example, the complementary nucleotides may be contiguous to each other or interspersed with non-complementary nucleotides.

Nucleic Acid Polymerase

In some embodiments, the nucleic acid polymerase may be selected from the family of DNA polymerases like E. coli DNA polymerase I (such as E. coli DNA polymerase I, Taq DNA polymerase, Tth DNA polymerase, TfI DNA polymerase and others). This polymerase may contain the naturally occurring wild-type sequences or modified variants and fragments thereof.

In some embodiments, the nucleic add polymerase may be selected from modified DNA polymerases of the family of DNA polymerases like E. coli DNA polymerase I, e.g., N-terminal deletions of the DNA polymerases, such as Klenow fragment of E. coli DNA polymerase I, N-terminal deletions of Taq polymerase (including the Stoffel fragment of Taq DNApolymerase, Klentaq-235, and Klentaq-278) and others.

In some embodiments, the nucleic acid polymerase includes, but is not limited to, thermostable DNA polymerases. Examples of thermostable DNA polymerases include, but are not limited to: Tth DNA polymerase, TfI DNA polymerase, Taq DNA polymerase, N-terminal deletions of Taq polymerase (e.g., Stoffel fragment of DNA polymerase, Klentaq-235, and Klentaq-278). Other DNA polymerases include KlenTaqi, Taquenase™ (Amersham), Ad-vanlaq™ (Clontech), GoTaq, GoTaq Flexi (Promega), and KlenTaq-S DNA polymerase.

In some embodiments, the nucleic acid polymerase may be commercially available DNA polymerase mixtures, including but are not limited to, TaqLA, TthLA or Expand High Fidelitypius Enzyme Blend (Roche); TthXL Klen TaqLA (Perkin-Elmer); ExTaq® (Takara Shuzo); Elongase® (Life Technologies); Advantage™ KlenTaq, Advantage™ Tth and Advantage2™ (Clontech); TaqExtender™ (Stratagene); Expand™ Long Template and Expand™ High Fidelity (Boehringer Mannheim); and TripleMaster™ Enzyme Mix (Eppendorf).

For further decreasing the amplification of undesired nucleic acid, one or more additional polymerase can be added into the reaction mixture. In some embodiments, the reaction mixture comprises high fidelity polymerase. In some embodiments, the high fidelity polymerase is PFU DNA Polymerase, Klentaq-1, Vent, or Deep Vent.

De-Blocking Agent

De-blocking agent can be selected according to the blocking group contained in the blocking primer. De-blocking agent can be any agent that may result in de-blocking the block group in the blocking primer under the condition of amplifying the target nucleic acid, when the nucleotide with the blocking group in the blocking primer is complementary to the corresponding nucleotide in the target nucleic acid. In some embodiments, the de-blocking agent is pyrophosphate, CS5 DNA polymerase with the mutations selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G or the combination thereof. In some embodiments, the de-blocking agent is ampliTaq or KlenTaq polymerase with F667Y mutation, or RNase H2.

In some embodiments, the de-blocking agent is pyrophosphate, when the blocking group is 2′, 3′-dideoxynucleotide. In some embodiments, the de-blocking agent is CS5 DNA polymerase with the mutations selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G or the combination thereof (e.g., those DNA polymerases shown in U.S. 20070154914), when the blocking group is 2′-O—PO₃ nucleotide. In some embodiments, the blocking group is 2′-O—PO₃ nucleotide and the de-blocking agent is ampliTaq or KlenTaq polymerase with F667Y mutation, when the blocking group is 2′-O—PO₃ nucleotide. In some embodiments, the de-blocking agent is RNase H2, when the blocking group is ribonucleotide residue.

Step of Incubating the Reaction Mixture Under a Condition for Amplification of the Target Nucleic Acid

Incubation of the reaction mixture of the present disclosure can be conducted in a multi-cycle process employing several alternating heating and cooling steps to amplify the DNA (see U.S. Pat. Nos. 4,683,202 and 4,683,195). In some embodiments, the incubation comprises the steps of denaturing the target nucleic acid; annealing the primers with the target nucleic acid to allow the formation of a target nucleic acid-primer hybrid; and incubating the target nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid.

An example of amplification process is briefly described below. First, a reaction mixture is heated to a temperature sufficient to denature the double stranded target DNA into its two single strands. The temperature of the reaction mixture is then decreased to allow specific single stranded primers to anneal to their respective complementary single-stranded target DNA. Following the annealing step, the temperature is maintained or adjusted to a temperature optimum of the DNA polymerase being used, which allows incorporation of complementary nucleotides at the 3′ ends of the annealed oligonucleotide primers thereby recreating double stranded target DNA. Using a heat-stable DNA polymerase, the cycle of denaturing, annealing and extension may be repeated as many times as necessary to generate a desired product, without the addition of polymerase after each heat denaturation (see “Current Protocols in Molecular Biology”, F. M. Ausubel et al., John Wiley and Sons, Inc., 1998).

In some embodiments, denaturing the target nucleic acid is conducted at about 90° C.-100° C. for from about 10 seconds to 10 minutes, preferably for the first circle for from about 1 to 8 minutes. In some embodiments, annealing the primers with the target nucleic acid is conducted at about 5° C.-60° C. for from about 3 seconds to 10 minutes. In some embodiments, incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid is conducted at about 60° C.-90° C. for from about 1 minute to 15 minutes.

In some embodiments, the incubation step is repeated at least 1 time, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times or 40 times. In some embodiments, the incubation step is repeated from about 20 times to about 50 times.

In some embodiments, the nucleic acid other than the target nucleic acid is not amplified in step (b) substantially. In some embodiments, the molar percentage of undesired nucleic acid in the product obtained after the incubation step is less than 20%, 15%, 10%, 5%, 3%, 2% or 1%.

The amplification method of the present disclosure can be used to construct DNA sequencing library. In some embodiments, the product obtained from the incubation step can be used as DNA sequencing library directly without enzyme digestion to reduce undesired amplification product. In some embodiments, the product obtained from the incubation step can be used as DNA sequencing library after the ligation of adaptor tags, but without enzyme digestion to reduce undesired amplification product.

“DNA sequencing library” as described in the present disclosure refers to a collection of DNA segments, in an abundance that can be sequenced, wherein one end or both ends of each segment in the collection of DNA segments contains a specific sequence partly or completely complementary to the primers used in sequencing, and thereby can be directly used in the subsequent DNA sequencing.

Some examples for construction of DNA sequencing library are shown in FIGS. 1-4 and 6-7.

In some embodiments, the method is used for selective enrichment of mutant nucleic acid in a sample comprising wildtype and mutant nucleic acid.

Some examples for selective enrichment of mutant nucleic acid are shown in FIG. 5-7.

Another aspect of the present disclosure provides method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least 20 different pairs of primers, wherein at least one primer of each primer pair is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension (“blocking primer”), (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polym erase; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; (c) determining the sequence of the products obtained from step (b).

The terms “sequencing” as used in the present disclosure refers to any and all biochemical methods that may be used to determine the identity and order of nucleotide bases including but not limited to adenine, guanine, cytosine and thymine, in one or more molecules of DNA. In some embodiments, the method is use for sequencing by capillary electrophoresis, PCR or high throughput sequencing (e.g., next-generation sequencing (NGS)).

Yet another aspect of the present disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least 20 different pairs of primers, wherein at least one primer of each primer pairs is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension (“blocking primer”), (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; (c) adding adaptor tag, molecular index tag and/or sample index tag to the product obtained from step (b); (d) determining the sequence of the products obtained from step (c).

In some embodiments, in the step (c), adaptor tag, molecular index tag and/or sample index tag is attached to the target nucleic acid obtained from step (b). The adaptor tag, molecular index tag and/or sample index tag can be attached according to the method mentioned above.

Yet another aspect of the present disclosure provides a kit of amplifying a target nucleic acid, wherein the kit comprises: (i) at least 20 different pairs of primers, wherein at least one primer of each primer pairs is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension, (ii) nucleic acid polymerase, and (iii) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase.

In some embodiments, the kit further comprises one or more agents selected from dNTPs, Mg²⁺ (e.g., MgCl₂), Bovin Serum Albumin, pH butler (e.g., Tris HCL), glycerol, DNase inhibitor, RNase, SO42⁻, Cl⁻, K⁺, Ca²⁺, Na⁺, and (NH₄)⁺.

In some embodiments, the kit further comprises an instruction showing how to conduct the amplification of the target nucleic acid (such as showing those methods of the present disclosure).

Yet another aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least one primer that is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension (“blocking primer”), wherein the blocking primer is modified so as to decrease the amplification of undesired nucleic acid, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid.

Yet another aspect of the present disclosure provides a kit of amplifying a target nucleic acid, wherein the kit comprises: (i) at least one primer that is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension (“blocking primer”), wherein the blocking primer is modified so as to decrease the amplification of undesired nucleic acid, (ii) nucleic acid polymerase, and (iii) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase.

Any embodiment following any aspect of the present disclosure can be applied to other aspects of the present disclosure, as long as the resulted embodiments are possible or reasonable for a person skilled in the art.

It is understood that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a “bridge probe” is a reference to one or more bridge probes, and includes equivalents thereof known to those skilled in the art and so forth.

All publications and patents cited in this specification are herein incorporated by reference to their entirety.

EXAMPLES

The invention will be more readily understood with reference to the following examples, which are not to be interpreted in any way as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.

Example 1. Multiplex PCR Amplification of Genomic DNA Target

A multiplex polymerase chain reaction was performed to selectively amplify 196 amplicons (the products amplified from target nucleic acid regions) across human genomic DNA. Each primer pair contains two primers with dideoxynucleoside terminated at its 3′ end and can selectively hybridize target nucleic acid. The sequence of each primer pair is shown in Table 3. The boldfaced sequences in each primer are the sequences for the following step of the library construction and other sequences in each primer are the sequences for selectively hybridizing target nucleic acid.

TABLE 3 Amplicons and corresponding primer pairs Forward_ Reverse_ Assay_ Target_ Primer_ Forward_  Primer_ Reverse_ ID Gene ID Primer ID Primer_ Chr 1 PDGFR IF ACACTCTTTCCCT 1R GTGACTGGAGT 4 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTAAAACA TCTTCCGATCTCA AGCTCTCATGTCT TGTGGTTGTGAA GAACT AACTGTTCAA 2 CDKN2 2F ACACTCTTTCCCT 2R GTGACTGGAGT 9 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTCATGGT TCTTCCGATCTC TACTGCCTCTGGT ACCAGCGTGTCC G AGGAA 3 SMAR 3F ACACTCTTTCCCT 3R GTGACTGGAGT 22 CB1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACATGG TCTTCCGATCTG AGATCGATGGGC CTGCCTGTCAGG A CAGAT 4 TP53 4F ACACTCTTTCCCT 4R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCBGAGG TCTTCCGATCTG TCACTCACCTGG GGGAGAAGTAA GTATATACacagt 5 RB1 5F ACACTCTTTCCCT 5R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGAACA TCTTCCGATCTAT AAACCATGTAATA TGTAACAGCATA AAATTCTGA CAAGGATCTTCC 6 SMAD 6F ACACTCTTTCCCT 6R GTGACTGGAGT 18 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCATTT TCTTCCGATCTG GTTTTCCCCTTTAA AGTAATGGTAGG ACAATTA TAATCTGTTTCTT AC 7 ATM 7F ACACTCTTTCCCT 7R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGAGGG TCTTCCGATCTA TACCAGAGACAGT ATTTTTATGTACT TTTCATTCCCTGA A 8 RB1 8F ACACTCTTTCCCT 8R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTYTGTTAT TCTTCCGATCTCT TTAGTTTTGAAAC CCACACACTCCA ACAGAGAA GTTAGGTA 9 ATM 9F ACACTCTTTCCCT 9R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCTATGC TCTTCCGATCTG AAGATACACAGTA TGCACTGAAAGA AAGGTTC GGATCGT 10 KDR 10F ACACTCTTTCCCT 10R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCTGA TCTTCCGATCTG CAAGAGCATGCCA GTTTCAGATCCA TAG CAGGGATTG 11 JAK3 11F ACACTCTTTCCCT HR GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCACCT TCTTCCGATCTG GATTGCATGCCA GCACTTCTCCAG CCCAA 12 PIK3CA 12F ACACTCTTTCCCT 12R GTGACTGGAGT 3 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGACRA TCTTCCGATCTA AGAACAGCTCAA CTGAATTTGGCT AGC GATCTCAGC 13 NPM1 13F ACACTCTTTCCCT 13R GTGACTGGAGT 5 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATGTCTA TCTTCCGATCTA TGAAGTGTTGTGG AAATTTTCCGTC TTCC TTATTTCATTTCT GT 14 RET 14F ACACTCTTTCCCT 14R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGGTCNG TCTTCCGATCTA ATTCCAGTTAAAT CGCAAAGTGATG GG TGTAAGTGTG 15 FGFR1 15F ACACTCTTTCCCT 15R GTGACTGGAGT 8 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACCCTG TCTTCCGATCTG CTTGCAGGATGG CAGTGATGGGTT GTAAACCTC 16 FLT3 16F ACACTCTTTCCCT 16R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATTTTC TCTTCCGATCTG GTGGAAGTGGGT CTTCCCAGCTGG TACC GTCAT 17 RB1 17F ACACTCTTTCCCT 17R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGTGGT TCTTCCGATCTA TTTAATTTCATCAT CTGCAGCAGATA GTTTCATA TGTAAGCAAAA 18 MLH1 18F ACACTCTTTCCCT 18R GTGACTGGAGT 3 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAAGTAG TCTTCCGATCTA TGATAAGGTCTAT GACAGATATTTC GCCCA TAGTGGCAGGG 19 SMAD 19F ACACTCTTTCCCT 19R GTGACTGGAGT 18 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCTGTT TCTTCCGATCTTT CACAATGAGCTTG TCCTGTATTTAGA CA TTGATTTAGTGG T 20 CDH1 20F ACACTCTTTCCCT 20R GTGACTGGAGT 16 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCGACA TCTTCCGATCTG CCCGATTCAAAGT GTTTCATAACCC G ACAGATCCAT 21 ATM 21F ACACTCTTTCCCT 21R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGCYTT TCTTCCGATCTTT CTGGCTGGATTTA TTTGGTTTTTAA AAT AATTAATGTTGG CA 22 PTEN 22F ACACTCTTTCCCT 22R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCRTGC TCTTCCGATCTT AGATAATGACAAG GACTTGTATGTAT GAA GTGATGTGTG 23 AKT1 23F ACACTCTTTCCCT 23R GTGACTGGAGT 14 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCGCCAC TCTTCCGATCTG AGAGAAGTTGTT TGAGAGCCACG GAG CACACT 24 FGFR3 24F ACACTCTTTCCCT 24R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTgCCCCT TCTTCCGATCTG GAGCGTCATCTG AGTTCCACTGCA AGGTGT 25 RET 25F ACACTCTTTCCCT 25R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCTGT TCTTCCGATCTC GCTGCATTTCAGA CACCCACATGTC GA ATCAAAT 26 ATM 26F ACACTCTTTCCCT 26R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGATGAG TCTTCCGATCTTC AAAYTCTCAGGAA AGGAAGTCACT ACTCTGT GATGTGAAG 27 FLT3 27F ACACTCTTTCCCT 27R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCCATT TCTTCCGATCTA CTTACCAAACTCT CCTAAATTGCTT AAATTTTC CAGAGATGAAA 28 KRAS 28F ACACTCTTTCCCT 28R GTGACTGGAGT 12 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCAGAA TCTTCCGATCTTT AACAGATCTGTAT CCTACTAGGACC TTATTTCA ATAGGTACA 29 STK11 29F ACACTCTTTCCCT 29R GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTGCCC TCTTCCGATCTCA GCAGGTACTTCT TTGTGCACAAGG ACATCAAG 30 FLT3 30F ACACTCTTTCCCT 30R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGGGTA TCTTCCGATCTTA TCCATCCGAGAAA GAAAAGAACGT CA GTGAAATAAGCT 31 ABL1 31F ACACTCTTTCCCT 31R GTGACTGGAGT 9 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCAACA TCTTCCGATCTG AGCCCACTGTCTA AAGAAATACAGC TG CTGACGGTG 32 VHL 32F ACACTCTTTCCCT 32R GTGACTGGAGT 3 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCCAGG TCTTCCGATCTG TCATCTTCTGCAAT GCATCCACAGCT C ACCGA 33 ATM 33F ACACTCTTTCCCT 33R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAAAGAT TCTTCCGATCTT CACCTTCAGAAGT GTTACCATTTTCT CACAG CATTCAGTGTCA T 34 KDR 34F ACACTCTTTCCCT 34R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATTTTAT TCTTCCGATCTG TTCCTCCCTGGAA TCAAGAGTAAG GTCC GAAAAGATTCAG ACT 35 FGFR2 35F ACACTCTTTCCCT 35R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCACCA TCTTCCGATCTTC TCCTGTGTGCAGG TCCATCTCTGAC ACCAGA 36 NRAS 36F ACACTCTTTCCCT 36R GTGACTGGAGT 1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCATGTAT TCTTCCGATCTTT TGGTCTCTCATGG CAATTTTTATTAA CAC AAACCACAGGG A 37 ERB34 37F ACACTCTTTCCCT 37R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACCAGT TCTTCCGATCTA GACTAGAAAGATC GAAACAAGACT AAATTCC CAGAGTTAGGG G 38 RB1 38F ACACTCTTTCCCT 38R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGACAT TCTTCCGATCTA GTAAAGGATAATT AAGATCTAGATG GTCAGTGAC CAAGATTATTTTT GG 39 SMO 39F ACACTCTTTCCCT 39R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTGCAG TCTTCCGATCTC AACATCAAGTTCA AGGACATGCAC ACAGT AGCTACATC 40 PIK3C 40F ACACTCTTTCCCT 40R GTGACTGGAGT 3 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTTAGGTG TCTTCCGATCTG GAATGAATGGCTG AAAGGGTGCTA AATTA AAGAGGTAAAG 41 KRAS 41F ACACTCTTTCCCT 41R GTGACTGGAGT 12 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCGTC TCTTCCGATCTC CACAAAATGATTC AGTCATTTTCAG TGAATTAG CAGGCCTTATA 42 SMAD 42F ACACTCTTTCCCT 42.R GTGACTGGAGT 18 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGGYGT TCTTCCGATCTT TCCATTGCTTACTT GTCCACAGGAC T AGAAGC 43 PIK3C 43F ACACTCTTTCCCT 43R GTGACTGGAGT 3 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCCATA TCTTCCGATCTT CTACTCATGAGGT GAAAGACGATG GTTTATTC GACAAGTAATGG 44 RB1 44F ACACTCTTTCCCT 44R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGAAG TCTTCCGATCTA GCAACTTGACAA ATAATTGAAGAA GAGAAAT ATTCATTCATGTG CA 45 CSF1R 45F ACACTCTTTCCCT 45R GTGACTGGAGT 5 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCTGC TCTTCCGATCTC TCAGAGCTCAAGT CTGAGCAGCTAT TC GTCACAG 46 PTEN 46F ACACTCTTTCCCT 46R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTGGTA TCTTCCGATCTG TGTATTTAACCATG TGAAGATATATTC CAGATCC CTCCAATTCAGG AC 47 ATM 47F ACACTCTTTCCCT 47R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGTTGG TCTTCCGATCTG AAGCTGCTTGGG TTATTTGAAGATA AAGAACTTCRGT GG 48 KDR 48F ACACTCTTTCCCT 48R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCTGAG TCTTCCGATCTC CATTAGCTTGCAA CTCTTTCTTCCTG GA AATGCTGAAA 49 GNAS 49F ACACTCTTTCCCT 49R GTGACTGGAGT 20 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCAGG TCTTCCGATCTC ACCTGCTTCGCT CAGTAAGCCAAC TGTTACCTTTT 50 PIK3C 50F ACACTCTTTCCCT 50R GTGACTGGAGT 3 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGCACA TCTTCCGATCTTC ATAAAACAGTTAG TCAAACAGGAG CCAGA AAGAAGGATGA 51 RB1 51F ACACTCTTTCCCT 51R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCTGCA TCTTCCGATCTT TTGGTGCTAAAAG GTAATAATTAAAT TTTCT TGGCATTCCTTT GG 52 MPL 52F ACACTCTTTCCCT 52R GTGACTGGAGT 1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCATCTA TCTTCCGATCTG GTGCTGGGCCTCA ACCAGGTGGAG CCGAAG 53 STK11 53F ACACTCTTTCCCT 53R GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGCATA TCTTCCGATCTGT GCCAGGGCATTG AGGCACGTGCTA GGGG 54 FGFR1 54F ACACTCTTTCCCT 54R GTGACTGGAGT 8 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTGTTT TCTTCCGATCTCT CTTTCTCCTCTGA AGTGCAGTTCCA AGAGG GATGAACAC 55 TP53 55F ACACTCTTTCCCT 55R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGAAAAT TCTTCCGATCTG GTTTCCTGACTCA TGACCCGGAAG GAGGG GCAGTC 56 RB1 56F ACACTCTTTCCCT 56R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTTCCTT TCTTCCGATCTT TGTAGTGTCCATA GTTGAAGAAGTA AATTCTTT TGATGTATTGTTT GC 57 CDH1 57F ACACTCTTTCCCT 57R GTGACTGGAGT 16 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCGTCGT TCTTCCGATCTG AATCACCACACTG GGAGGCTGTATA AAAG CACCATATTGA 58 FLT3 58F ACACTCTTTCCCT 58R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCACAT TCTTCCGATCTCT TGCCCCTGACAAC TCACCACTTTCC CGTGG 59 PDGFR 59F ACACTCTTTCCCT 59R GTGACTGGAGT 4 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCCTGA TCTTCCGATCTA GTCATTTCTTCCTT CTATGTGTCGAA TTCC AGGCAGTGTA 60 HNF1A 60F ACACTCTTTCCCT 60R GTGACTGGAGT 12 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATGAGC TCTTCCGATCTC TACCAACCAAGAA AGATCCTGTTCC GG AGGCCTAT 61 MET 61F ACACTCTTTCCCT 61R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATTTTG TCTTCCGATCTG GTCTTGCCAGAGA CTTTGGAAAGTC CATG TGCAAACTCAA 62 MET 62F ACACTCTTTCCCT 62R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCCCT TCTTCCGATCTTC GCAACAGCTGAAT TCAATGGGCAAT C GAAAATGTA 63 MET 63F ACACTCTTTCCCT 63R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCAGTG TCTTCCGATCTCA CTAACCAAGTTCT TGGAGTATACTT TTCT TTGTGGTTTGC 64 AKT1 64F ACACTCTTTCCCT 64R GTGACTGGAGT 14 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACRATG TCTTCCGATCTC ACTTCCTTCTTGA CAGGATCACCTT GGA GCCGAA 65 GNA11 65F ACACTCTTTCCCT 65R GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTGTGT TCTTCCGATCTC CCTTTCAGGATGG CACTGCTTTGAG TG AACGTGAC 66 GNAS 66F ACACTCTTTCCCT 66R GTGACTGGAGT 20 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCYCCAC TCTTCCGATCTCT CAGCATGTTTGA TTGCTTCTGTGT TGTTAGGG 67 KIT 67F ACACTCTTTCCCT 67R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCTAG TCTTCCGATCTC TGCATTCAAGCAC CAATTTAAGGGG AATGG ATGTTTAGGCT 68 PTPN11 68F ACACTCTTTCCCT 68R GTGACTGGAGT 12 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCAATG TCTTCCGATCTG GACTATTTTAGAA GGCAATTAAAAG GAAATGGA AGAAGAATGGA 69 ALK 69F ACACTCTTTCCCT 69R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTCTCT TCTTCCGATCTG CGGAGGAAGGAC CAGAGAGGGAT TT GTAACCAAAATT 70 JAK3 70F ACACTCTTTCCCT 70R GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGACCT TCTTCCGATCTC TAGCAGGATCCAG CTGTCGGTGAGC G ACTGA 71 NRAS 71F ACACTCTTTCCCT 71R GTGACTGGAGT 1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGAAAG TCTTCCGATCTC CTGTACCATACCT CAGTTCGTGGGC GTCT TTGTT 72 BRAF 72F ACACTCTTTCCCT 72R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTCACA TCTTCCGATCTTC ATGTCACCACATT TACCAAGTGTTT ACATACT TCTTGATAAAAA C 73 ATM 73F ACACTCTTTCCCT 73R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATTTGA TCTTCCGATCTG CCGTGGAGAAGT AGAGAGCCAAA AGAATC GTACCATAGGTA 74 KRAS 74F ACACTCTTTCCCT 74R GTGACTGGAGT 12 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCATGT TCTTCCGATCTC ACTGGTCCCTCAT CAAGAGACAGG TGC TTTCTCCATCA 75 MET 75F ACACTCTTTCCCT 75R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATGATA TCTTCCGATCTC GCCGTCTTTAACA AGAAATGGTTTC AGCTC AAATGAATCTGT 76 EGFR 76F ACACTCTTTCCCT 76R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCAGGA TCTTCCGATCTCA ACGTACTGGTGAA TTTTCCTGACAC AAC CAGGGAC 77 TP53 77F ACACTCTTTCCCT 77R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGTCCC TCTTCCGATCTG AGAATGCAAGAA GAGCAGCCTCTG GC GCATT 78 SMO 78F ACACTCTTTCCCT 78R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGTTTT TCTTCCGATCTG GTGGGCTACAAG GGCACTTGCTGC AACT CAGTA 79 FBXW7 79F ACACTCTTTCCCT 79R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTAAACTT TCTTCCGATCTG ACTTTGCCTGTGA CACCTATAAGAA CTGC AGATGTGCAGA 80 SMO 80F ACACTCTTTCCCT 80R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTGGAG TCTTCCGATCTCT AAGATCAACCTGT CACCCTCAGCCT TTGC TGGG 81 MET 81F ACACTCTTTCCCT 81R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCCTGA TCTTCCGATCTG ATGATGACATTCT TCAACAAAAACA TTTCG ATGTGAGATGTC 82 ATM 82F ACACTCTTTCCCT 82R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACCAGA TCTTCCGATCTG GTTTCAACAAAGT AGTGGAAGAAG AGCTG GCACTGTG 83 FGFR2 83F ACACTCTTTCCCT 83R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCGGCAY TCTTCCGATCTA AGGATGACTGTTA GAGTTAGCACAC C CAGACTG 84 PTEN 84F ACACTCTTTCCCT 84R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATTTCCA TCTTCCGATCTA TCCTGCAGAAGAA GGATGGATTCGA GC CTTAGACTTGA 85 VHL 85F ACACTCTTTCCCT 85R GTGACTGGAGT 3 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCTCTT TCTTCCGATCTC TAACAACCTTTGC AATATCACACTG TTGTC CCAGGTACTG 86 KIT 86F ACACTCTTTCCCT 86R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCTCTTC TCTTCCGATCTG CATTGTAGAGCAA TTCTCTCTCCAG ATCC AGTGCTCTAAT 87 FBXW 87F ACACTCTTTCCCT 87R GTGACTGGAGT 4 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTATTCAA TCTTCCGATCTG ATAACACCCAATG GTTCACAACTAT AAGAATGT CAATGAGTTCAT 88 EGFR 88F ACACTCTTTCCCT 8SR GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCATC TCTTCCGATCTG ACGCAGCTCATGC AGATAAGGAGC CAGGATCCTC 89 SMO 89F ACACTCTTTCCCT 89R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCCAGC TCTTCCGATCTG ATGTCACCAAGAT CTTCTGGGACTG G GAGTACAG 90 FBXW 90F ACACTCTTTCCCT 90R GTGACTGGAGT 4 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAAGTCC TCTTCCGATCTG CAACCATGACAAG TGTCCGATCTGT ATTTT AGATCCACTAA 91 PTEN 91F ACACTCTTTCCCT 91R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGCCAG TCTTCCGATCTTT CTAAAGGTGAAGA TGTACTTTACTTT T CATTGGGAGA 92 SMAD 92F ACACTCTTTCCCT 92R GTGACTGGAGT 18 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCCAT TCTTCCGATCTCA CAAGTATGATGGT TCCAGCATCCAC GAAGG CAAGTAAT 93 PIK3C 93F ACACTCTTTCCCT 93R GTGACTGGAGT 3 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCTTCC TCTTCCGATCTG ACACAATTAAACA AATTGCACAATC GCATG CATGAACAGC 94 KIT 94F ACACTCTTTCCCT 94R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTAGTGT TCTTCCGATCTG ATTCACAGAGACT AAACGTGAGTAC TGGCA CCATTCTCTG 95 KIT 95F ACACTCTTTCCCT 95R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCATGTT TCTTCCGATCTT TCCAATTTTAGCG GTCCAAGCTGCC AGTGC TTTTATTGTC 96 ATM 96F ACACTCTTTCCCT 96R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGTGTA TCTTCCGATCTG GGAAAGGTACAAT TGGATTCCTCTA GATTTCC AGTGAAAATCAT GA 97 SMAD 97F ACACTCTTTCCCT 97R GTGACTGGAGT 18 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGAAGG TCTTCCGATCTA ACTGTTGCAGATA AAGTAGGCAGC GCATC CTTTATAAAAGC A 98 ALK 98F ACACTCTTTCCCT 98R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGGGTGA TCTTCCGATCTG GGCAGTCTTTACT GGAAGAAAGGA CA AATGCATTTCCT 99 EGFR 99F ACACTCTTTCCCT 99R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCGAAG TCTTCCGATCTG CCACACTGACGT CTGCCTCCTGGA CTATGTC 100 8RAF 100F ACACTCTTTCCCT 100R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTACCAT TCTTCCGATCTGT CCACAAAATGGAT AAGTAAAGGAA CCAG AACAGTAGATCT CA 101 ABL1 10IF ACACTCTTTCCCT 101R GTGACTGGAGT 9 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCGAAAC TCTTCCGATCTG TGCCTGGTAGGG GAGCCAAGTTCC CCATC 102 ERBB4 102F ACACTCTTTCCCT 102R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACTTAC TCTTCCGATCTTC GTGGACATTTCTT CACTGTCATTGA GACAC AATTCATGCA 103 ARC 103F ACACTCTTTCCCT 103R GTGACTGGAGT 5 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCAGACT TCTTCCGATCTC GCAGGGTTCTAGT CCACTCATGTTT TTATC AGCAGATGTAC 104 FGFR2 104F ACACTCTTTCCCT 104R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCAGTCC TCTTCCGATCTG GGCTTGGAGGAT GAGTGGGGATG GGAGAA 105 PTEN 105F ACACTCTTTCCCT 105R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACCACA TCTTCCGATCTT GCTAGAACTTATC GTGCATATTTATT AAACC ACATCGGGGC 106 RET 106F ACACTCTTTCCCT 106R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGGCTAT TCTTCCGATCTC GGCACCTGCAAC AGCCCCACAGA GGTCTC 107 ERBB4 107F ACACTCTTTCCCT 107R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTGCAG TCTTCCGATCTTT TCTTACATTTGACC TTCCTCCAAAGG ATGA TCATCAGTTC 108 CTNNB 108F ACACTCTTTCCCT 108R GTGACTGGAGT 3 1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTAGCTG TCTTCCGATCTGT ATTTGATGGAGTT AAAGGCAATCCT GGAC GAGGAAGAG 109 HNF1A 109F ACACTCTTTCCCT 109R GTGACTGGAGT 12 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTGGGC TCTTCCGATCTC TCCAACCTCGTC ACAAGCTGGCCA TGGAC 110 PDGFR 110F ACACTCTTTCCCT 110R GTGACTGGAGT 4 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTGGTA TCTTCCGATCTG ATTCACCAGTTAC CCTTATGACTCA CTGTC AGATGGGAGTT 111 STK11 111F ACACTCTTTCCCT 111R GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTGGGT TCTTCCGATCTG ATGGACACGTTCA CAAGGTGAAGG TC AGGTGC 112 ATM 112F ACACTCTTTCCCT 112R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCTGTT TCTTCCGATCTA CCTCAGTTTGTCA AGGTAATTTGCA CTAAA ATTAACTCTTGAT T 113 KDR 113F ACACTCTTTCCCT 113R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAACAAC TCTTCCGATCTG ACTTGAAAATCTG GTTTGCACTCCA AGCAG ATCTCTATCAG 114 ERBB2 114F ACACTCTTTCCCT 114R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAATTCC TCTTCCGATCTC AGTGGCCATCAAA ACCCTCTCCTGC GT TAGGA 115 FBXW 115F ACACTCTTTCCCT 115R GTGACTGGAGT 4 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACCYTG TCTTCCGATCTG CAATGTTTGTAAA TGTGAATGCAAT CACTG TCCCTGTC 116 SMAD 116F ACACTCTTTCCCT 116R GTGACTGGAGT IS 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCTGAT TCTTCCGATCTA GTCTTCCAAATCT AATTCACTTACA TTTCT CCGGGCC 117 SMAD 117F ACACTCTTTCCCT 117R GTGACTGGAGT 18 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTTGAT TCTTCCGATCTGT TTGCGTCAGTGTC AGGTGGAATAG AT CTCCAGC 118 EGFR 118F ACACTCTTTCCCT 118R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTAACG TCTTCCGATCTT TCTTCCTTCTCTCT GAGTTTCTGCTT CTGT TGCTGTGTG 119 JAK3 119F ACACTCTTTCCCT 119R GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCACT TCTTCCGATCTC GTCTCCAGCCATG AAATTTTGTGCT CACAGACCT 120 IDH1 120F ACACTCTTTCCCT 120R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTGCCA TCTTCCGATCTC ACATGACTTACTT CAGAATATTTCGT GATCC ATGGTGCCAT 121 ARC 121F ACACTCTTTCCCT 121R GTGACTGGAGT 5 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATGCCT TCTTCCGATCTTT CCAGTTCAGGAA ATTTCTGCCATG AAT CCAACA 122 PGFR2 122F ACACTCTTTCCCT 122R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAAGTCC TCTTCCGATCTG TCACCTTGAGAAC GGCTGGGCATC C ACTGTA 123 PTEN 123F ACACTCTTTCCCT 123R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGGACC TCTTCCGATCTA AGAGGAAACCTC AATGATCTTGAC AG AAAGCAAATAAA GAC 124 SMAD 124F ACACTCTTTCCCT 124R GTGACTGGAGT 18 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTGATC TCTTCCGATCTG TATGCCCGTCTCT AGTTGTATCACC GG TGGAATTGGTA 125 ARC 125F ACACTCTTTCCCT 125R. GTGACTGGAGT 5 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACTGAG TCTTCCGATCTA AGCACTGATGATA AATGTAAGCCAG AACAC TCTTTGTGTCA 126 TP53 126F ACACTCTTTCCCT 126R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTGTCC TCTTCCGATCTA TGCTTGCTTACCT CTACTCAGGATA C GGAAAAGAGAA 127 ERBB4 127F ACACTCTTTCCCT 127R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGTGGA TCTTCCGATCTCT TAACACATACCAG GGACATTTTTCC GTGA ACACAGTTTG 128 RB1 128F ACACTCTTTCCCT 128R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTGATT TCTTCCGATCTA TTCTAAAATAGCA AAATTTCAgccgg GGCTCTTAT gcgc 129 ATM 129F ACACTCTTTCCCT 129R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGCTTA TCTTCCGATCTC ATTATTCTGAAGG AGGTCTTCCAGA GCCG TGTGTAATACATT 130 HRAS 130F ACACTCTTTCCCT 130R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCGGTGC TCTTCCGATCTTC GCATGTACTGGT CAACAGGCACG TCTCC 131 PTPN1 131F ACACTCTTTCCCT 131R GTGACTGGAGT 12 1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCTTCAT TCTTCCGATCTAT GATGTTTCCTTCGT TGAAACACTACA AGG GCGCAGG 132 SMO 132F ACACTCTTTCCCT 132R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCCAA TCTTCCGATCTC TGAGACTCTGTCC GGGCAAGACCT TGC CCTACTT 133 KIT 133F ACACTCTTTCCCT 133R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCTCAA TCTTCCGATCTT CCATCTGTGAGTC GGACTTTTGAGA CA TCCTGGATGAA 134 ATM 134F ACACTCTTTCCCT 134R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCAGCTG TCTTCCGATCTA TTACCTGTTTGAA GATCCAATGCTG AAACATTT GCCTA 135 EGFR 135F ACACTCTTTCCCT 135R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCACTA TCTTCCGATCTG CATTGACGGCCC TGGAAAGTGAA GGAGAACAGAA C 136 NOTC 136F ACACTCTTTCCCT 136R GTGACTGGAGT 9 H1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGACCAG TCTTCCGATCTC CGAGGATGGCAG ACTCAGGAAGCT CCGGC 137 FGFR3 137F ACACTCTTTCCCT 137R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCAATGT TCTTCCGATCTG GCTGGTGACCGA GGTCATGCCAGT G AGGACG 138 FGFR3 138F ACACTCTTTCCCT 138R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGGGAC TCTTCCGATCTG GACTCCGTGTTTG TGAGGGGTCCCT AGCAG 139 KDR 139F ACACTCTTTCCCT 139R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCCACT TCTTCCGATCTG GGATGCTGCACA TTGACTGAACTT CCAAAGCAC 140 ABL1 140F ACACTCTTTCCCT 140R GTGACTGGAGT 9 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTCTTG TCTTCCGATCTTC TTGGCAGGGGTC ATCCACAGGTAG GGGC 141 APC 141F ACACTCTTTCCCT 141R GTGACTGGAGT 5 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCATCAG TCTTCCGATCTA CTGAAGATGAAAT GCACCCTAGAAC AGGATGTAA CAAATCC 142 TP53 142F ACACTCTTTCCCT 142R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTSCCAGT TCTTCCGATCTAT TGCAAACCAGAC CAGTGAGGAATC AGAGGC 143 FGFR3 143F ACACTCTTTCCCT 143R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTGTCT TCTTCCGATCTCA GTCCTGGGAGTCT TCCCTGTGGAGG AGCT 144 KIT 144F ACACTCTTTCCCT 144R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTGTTG TCTTCCGATCTA TGCTTCTATTACAG ATGATCCTTGCC GCTC AAAGACAACT 145 KDR 145F ACACTCTTTCCCT 145R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTGGCT TCTTCCGATCTC TTGAATCATTAGC GGACTCAGAAC GTTAC CACATCATAAAT 146 ERB84 146F ACACTCTTTCCCT 146R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAAGGTT TCTTCCGATCTA TACACATTTTAATC CATTCAGCAAAC CCATTTT AAGCTCAAAAC 147 ATM 147F ACACTCTTTCCCT 147R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATTAGG TCTTCCGATCTTA TGGACCACACAG AGGTGAGCCTTC GA CCTTC 148 RET 148F ACACTCTTTCCCT 148R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTACTCGT TCTTCCGATCTTA GCTATTTTTCCTCA CGTGAAGAGGA CAG GCCAG 149 IDH2 149F ACACTCTTTCCCT 149R GTGACTGGAGT 15 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCCTAC TCTTCCGATCTCA CTGGTCGCCATG TTGGGACTTTTC CACATCTTCT 150 MET 150F ACACTCTTTCCCT 150R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGACATG TCTTCCGATCTCT TCTTTCCCCACAAT TTCATCTGTAAA CATA GGACCGGTTC 151 SMAR 151F ACACTCTTTCCCT 151R GTGACTGGAGT 22 CB1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTACTCA TCTTCCGATCTC TAGGTGGGAAACT CTAACACTAAGG ACCTC GTGCGT 152 SRC 152F ACACTCTTTCCCT 152R GTGACTGGAGT 20 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTTCCT TCTTCCGATCTC GGAGGACTACTTC TCTGCCTGCCTG ACG CTGTT 153 ATM 153F ACACTCTTTCCCT 153R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATCTAG TCTTCCGATCTTC GATCCAAATTTTA ATCTTGTACTGG GAAGTCAAG AGAAAATTCTTG TG 154 NOTC 154F ACACTCTTTCCCT 154R GTGACTGGAGT 9 H1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCACTGC TCTTCCGATCTT CGGTTGTCAATCT GACGCCACAGTC C AGGAC 155 KIT 155F ACACTCTTTCCCT 155R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCACCT TCTTCCGATCTA TCTTTCTAACCTT AACGTGATTCAT TTCTTATGT TTATTTGTTCAA AGC 156 KDR 156F ACACTCTTTCCCT 156R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGATGCT TCTTCCGATCTA CACTGTGTGTTGC ATAATTGGGGTC T CCTCCCT 157 RET 157F ACACTCTTTCCCT 157R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCGCAGC TCTTCCGATCTT CTGTACCCAGTG GCTACCACAAGT TTGCCC 158 PTEN 158F ACACTCTTTCCCT 158R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGCFACG TCTTCCGATCTA ACCCAGTTACCAT GCTACCTGTTAA AGC AGAATCATCTGG A 159 GNAQ 159F ACACTCTTTCCCT 159R GTGACTGGAGT 9 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGAGGTG TCTTCCGATCTA ACATTTTCAAAGC AATATAGCACTA AGTG CTTACAAACTTA GGG 160 ERB84 160F ACACTCTTTCCCT 160R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAAAGTG TCTTCCGATCTG GCTAAAGTTGATC TCCTGAGCAGC TGATTGT MTCCAG 161 JAK2 161F ACACTCTTTCCCT 161R GTGACTGGAGT 9 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCTTAG TCTTCCGATCTC TCTTTCTTTGAAG CTTTCTCAGAGC CAGCA ATCTGTTTTTG 162 ERBB4 162F ACACTCTTTCCCT 162R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATAACT TCTTCCGATCTT CATTCATCGCCAC GAATGGTGTCTG ATAGG CATAACAAAGG 163 EGFR 163F ACACTCTTTCCCT 163R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTCTCT TCTTCCGATCTT GTGTTCTTGTCCC GTATAAGGTAAG C GTCCCTGG 164 CSF1R 164F ACACTCTTTCCCT 164R GTGACTGGAGT 5 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCATCCAT TCTTCCGATCTTC GGAGGAGTTGAA AGGTGCTCACTA GTTT GAGCTC 165 VHL 165F ACACTCTTTCCCT 165R GTGACTGGAGT 3 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCTTG TCTTCCGATCTA TTCGTTCCTTGTAC GGAGACTGGAC TGAG ATCGTCAG 166 PDGFR 166F ACACTCTTTCCCT 166R GTGACTGGAGT 4 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGGCVC TCTTCCGATCTC CATTTACATCATCA ACCCAGAGAAG CCAAAGAAAG 167 ATM 167F ACACTCTTTCCCT 167R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATAGGA TCTTCCGATCTC AGTAGAGGAAAG CAGGTACAGTAA TATTCTTCAG GTAGGTCATGT 168 SMAR 168F ACACTCTTTCCCT 168R GTGACTGGAGT 22 CB1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCACCCC TCTTCCGATCTCT TACACTTGGCTG GGTAACCAGCCC ATCAG 169 STK11 169F ACACTCTTTCCCT 169R GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCCCC TCTTCCGATCTG TCGAAATGAAGCT GGAGCCTCATCC A CTCTG 170 HRAS 170F ACACTCTTTCCCT 170R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCAGGC TCTTCCGATCTC TCAC.CTCTATAGTG ACCACCAGCTTA G TATTCCGT 171 ERB82 171F ACACTCTTTCCCT 171R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCCTCTC TCTTCCGATCTG AGCGTACCCTTGT GTGCAGCTGGT GACACA 172 EZH2 172F ACACTCTTTCCCT 172R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTATACAAT TCTTCCGATCTG GCCACCTGAATAC TGCCAGCAATAG AGG ATGCTAGA 173 FBXW 173F ACACTCTTTCCCT 173R GTGACTGGAGT 4 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTCCTG TCTTCCGATCTT CCATCATATTGAAC GCAGAGGGAGA ACAG AACAGAAAAAC 174 KIT 174F ACACTCTTTCCCT 174R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTAGAG TCTTCCGATCTAT CATGACCCATGAG GGACATGAAACC TG TGGAGTT 175 RB1 175F ACACTCTTTCCCT 175R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGTTCTT TCTTCCGATCTC CCTCAGACATTCA CAGGGTAGGTC AACGT AAAAGTATCCTT 176 EGFR 176F ACACTCTTTCCCT 176R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTACCAG TCTTCCGATCTG ATGGATGTGAACC GAGTATCCCATC CC TTGGAGAGTC 177 ERBB4 177F ACACTCTTTCCCT 177R GTGACTGGAGT 2 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTGCCA TCTTCCGATCTTT TTTTGGATATATTC GTCCCACGAATA CTTACCT ATGCGTAAAT 178 CDH1 178F ACACTCTTTCCCT 178R GTGACTGGAGT 16 ACACGACGCTCTT TCAGACGTGTGC CCGATCFACTTGG TCTTCCGATCTTC TTGTGTCGATCTCT TTCAATCCCACC CT ACGGTAAT 179 STK11 179F ACACTCTTTCCCT 179R GTGACTGGAGT 19 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGACAC TCTTCCGATCTA CAAGGACCGGTG CATCGAGGATGA CATCATCTACA 180 ABL1 180F ACACTCTTTCCCT 180R GTGACTGGAGT 9 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCAAGT TCTTCCGATCTT ACTTACCCACTGA GCAGCTCCTTGG AAAGC TGAGTAA 181 PTEN 181F ACACTCTTTCCCT 181R GTGACTGGAGT 10 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGCTCA TCTTCCGATCTT TTTTTGTTAATGGT GCTTGCAAATAT GGCT CTTCTAAAACAA CTA 182 ATM 182F ACACTCTTTCCCT 182R GTGACTGGAGT 11 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTGATAA TCTTCCGATCTCA ATKAGCAGTCAGC TGGAATGTTGTT AGAA TGCCTACC 183 RB1 183F ACACTCTTTCCCT 183R GTGACTGGAGT 13 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTTCTTA TCTTCCGATCTC TTCCCACAGTGTA CTGCAGAATGAG TCGG TATGAACTCAT 184 KDR 184F ACACTCTTTCCCT 184R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGCFTT TCTTCCGATCTC AAAAGTTCTGCFT ACCATTCCACTG CCTCA CAGAAGAAAT 185 EGFR 185F ACACTCTTTCCCT 185R GTGACTGGAGT 7 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCCTC TCTTCCGATCTA AAAAGAGAAATC AATATGTACTACG ACGCAT AAAATTCCTATG CC 186 NOTC 186F ACACTCTTTCCCT 186R GTGACTGGAGT 9 H1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAAGATC TCTTCCGATCTC ATCTGCTGGCCGT CAGCCTCTCGGG TACAT 187 TP53 187F ACACTCTTTCCCT 187R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGAGG TCTTCCGATCTA CAAGCAGAGGCT CCTAGGAGATAA G CACAGGCC 188 APC 188F ACACTCTTTCCCT 188R GTGACTGGAGT 5 ACACGACGCTCTT TCAGACGTGTGC CCGATCTAGAGAA TCTTCCGATCTA CGCGGAATTGGTC GCCATTCATACC T TCTCAGGAA 189 SMAD4 189F ACACTCTTTCCCT 189R GTGACTGGAGT 18 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCTTTTTY TCTTCCGATCTC CTTCCTAAGGTTG GTGCACCTGGA CACA GATGCT 190 SMAR 190F ACACTCTTTCCCT 190R GTGACTGGAGT 22 CB1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTCTTGTA TCTTCCGATCTA TCTCCTCAGGGAA GACAAGAAGAG CAG AACCTTCCCC 191 ERB82 191F ACACTCTTTCCCT 191R GTGACTGGAGT 17 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTACATG TCTTCCGATCTG GGTGCTTCCCATT GGGCAAGGTTA C GGTGAAG 192 NRAS 192F ACACTCTTTCCCT 192R GTGACTGGAGT 1 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTACAAA TCTTCCGATCTG GTGGTTCTGGATT CGAGCCACATCT AGCTG ACAGTACTTTA 193 FGFR3 193F ACACTCTTTCCCT 193R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTCATGTC TCTTCCGATCTC TTTGCAGCCGAG CAAGAAAGGCC G TGGGCT 194 KIT 194F ACACTCTTTCCCT 194R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTGGTGAT TCTTCCGATCTA CTATTTTTCCCTTT GAAACAGGCTG CTCCC AGTTTTGGTC 195 KDR 195F ACACTCTTTCCCT 195R GTGACTGGAGT 4 ACACGACGCTCTT TCAGACGTGTGC CCGATCTTAGACA TCTTCCGATCTT AGGTCTTCCTTCC CCTCCTCCATAC ACTT AGGAAACAG 196 PIK3C 196F ACACTCTTTCCCT 195R GTGACTGGAGT 3 A ACACGACGCTCTT TCAGACGTGTGC CCGATCTTTCTCA TCTTCCGATCTT ATGATGCTTGGCT GGCTGGACAAC C AAAAATGGA

Amplification of the target nucleic acid regions. A pool of 196 amplicon primer pairs in a concentration of 50 nM for each primer were added to a single PCR tube, 50 ng of human genomic DNA (GenBank No.: NA12878) and 10 of amplification reaction mixture which contains 3% glycerol, 0.2 nM dNTPs, 50nM pyrophosphate and 2 units of KlenTaq-S DNA polymerase to a final volume of 20 μL with DNase/RNase free water. The PCR tube was put on a thermal cycler and run the following temperature profile to get the amplified amplicon library. An initial holding stage was carried out at 98° C. for 2 minutes, followed by 98° C. 15 seconds, 55° C. 8 minutes, for 17 cycles. After cycling, the reaction was held at 72° C. for 5 minutes and then 4° C. until proceeding to the beads purification step to remove excess primers. The tube cap was carefully removed and 24 μL of Agencourt AMPure® XP Reagent (Beckman Coulter, CA) was added to the reaction mixture to purify the DNA. The reaction mixture was vortex mixed and incubated for 5 minutes at room temperature. The tube was placed in a magnetic rack and incubated until solution clears. The supernatant was carefully remove and discarded without disturbing the pellet, then 150 μL of freshly prepared 70% ethanol was added. The reaction mixture was vortexed to mix well, pulsed spin, and incubated until solution clears. The supernatant was carefully removed and discarded without disturbing the pellet. Another potion of 150 μL of freshly prepared 70% ethanol was added. The reaction mixture was vortexed to mix well, pulsed spin, and incubated until solution clears. The supernatant was carefully removed and discarded without disturbing the pellet. Leave the tube open to evaporate for about 2 minutes. The remaining bead pellet in the tube contains the purified DNA.

Construction of library. Then 50 μL of Platinum® PCR SuperMix High Fidelity DNA Polymerase (Thermo Fisher, Cat #12532016) and 2 μL of Library Amplification Barcoded Primer Mix (10 μM concentration for each primer was added to bead pellet). The sequence of each primer is shown in Table 4. The PCR tube was put on a thermal cycler and run the following temperature profile to get the amplified amplicon library. An initial holding stage was carried out at 98° C. for 2 minutes, followed by 98° C. 15 seconds, 60° C. 1 minute, for 5 cycles. After cycling, the reaction was held at 72° C. for 5 minutes and then kept at 4° C. until purification. The tube cap was carefully removed and 44 μL of Agencourt AMPure® XP Reagent (Beckman Coulter, CA) was added to the reaction mix for purifying the product. The reaction was vortex mixed and incubated for 5 minutes at room temperature. The tube was placed in a magnetic rack and incubated until solution clears. The supernatant was carefully removed and discarded without disturbing the pellet. Then 150 μL of freshly prepared 70% ethanol was added to the pellet; the reaction mixture was vortexed to mix well, pulsed spin, and incubated until solution clears; the supernatant was carefully removed and discarded without disturbing the pellet; the foregoing wash step was repeated for another time. After the wash, leave the tube open to evaporate for about 2 minutes. Then 50 μL of low TE buffer was added and the solution was vortexed thoroughly. The tube was placed in the magnet until solution clears. The supernatant containing library was collected to a separate clean tube. The library was quantified using Qubit® 2.0 Fluorometer (Life Technologies, CA) and Bioanalyzer (Agilent Technologies, CA) according to manufacturer protocol.

TABLE 4 Primers for construction of library Primer Name Primer Sequence R2TruSeqBC001 CAAGCAGAAGACGGCATACGAGATcgcgac tgaaGTGACTGGAGTTCAGACGTGT R2TruSeqBC002 CAAGCAGAAGACGGCATACGAGATagcatc gataGTGACTGGAGTTCAGACGTGT R2TruSeqBC003 CAAGCAGAAGACGGCATACGAGATcgacac atggGTGACTGGAGTTCAGACGTGT R2TruSeqBC004 CAAGCAGAAGACGGCATACGAGATcgacta cgcaGTGACTGGAGTTCAGACGTGT R2TruSeqBC005 CAAGCAGAAGACGGCATACGAGATcactgc tgagGTGACTGGAGTTCAGACGTGT R2TruSeqBC006 CAAGCAGAAGACGGCATACGAGATtcgctg tacaGTGACTGGAGTTCAGACGTGT R2TruSeqBC007 CAAGCAGAAGACGGCATACGAGATcgctgc agtaGTGACTGGAGTTCAGACGTGT R2TruSeqBC008 CAAGCAGAAGACGGCATACGAGATagactt gcagGTGACTGGAGTTCAGACGTGT R1_TruSeq_primer AATGATACGGCGACCACCGAGATCTACACT CTTTCCCTACACGAC

The library was sequenced on Illumina MiSeq sequencer according to manufacturer's procedure.

Data Processing

Sequencing reads were aligned to GRC37/hg19 reference genome downloaded from web of ucsc genome browser (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/) using the software of bowtie2 (downloaded from https://sourceforge.net/projects/bowtie-bio/files/bowtie/1.2.1.1) with default settings. The aligned reads were further assigned to amplicons based on the match between positions of reads of R1 and R2 in genome and positions of forward and reverse primers of designed assays. The preliminary results indicated that performances of cancer hot spot panel were (1) 69.7% reads aligned to genome; (2) 95.5% reads aligned to target regions of design; (3) 98.1% of assays with amplicon read coverage within 5-fold of the mean average.

Example 2. Multiplex Enrichment of Mutant Nucleic Acid for Sequencing

In 20 μL PCR reaction solution, two pools of 8 primer pairs (see Table 5) with each primer containing dideoxynucleotide at its 3′ end in 0.5 μM concentration were added together with 2 μL of 10X PCR buffer, 3 mM MgCl₂, 0.2 mM dNTP, 50 nM pyrophosphate, 2 units of AmpliTaq DNA polymerase and 1%, 0.1% or 0.01% mutant nucleic acid (Horizon discovery, Cambridge, United Kingdomnin), 30 ng of human genomic DNA (GenBank No.: NA12878). The PCR tube was loaded on a thermal cycler and run the following temperature profile: 95° C. for 2 min; 95° C. 15 seconds, 65° C. 120 seconds, for 40 cycles; hold at 4° C. 5 μL ExoSAP-IT™ solution (Affymetrix, CA) was added to the tube, and the reaction was incubated at 37° C. for 15 min, 80° C. for 10 min, held at 4° C. 2 μL of reaction solution was used to perform cycle sequencing with BigDye™ Terminator v3.1 cycle sequencing kit (Life Technologies, CA) and the resulting DNA was purified according to manufacturer protocol. The purified sample electrophoresis was carried out on ABI Prism 3730 DNA analyzer according to manufacturer recommended protocol. Electropherogram in FIG. 12 showed examples of enrichment of mutant nucleic acid (EGFR COSMIC6240 mutation, EGFR COSMIC6252mutation, COSMIC6241 mutation, COSMIC6224 mutation, COSMIC6223 mutation and COSMIC6213 mutation from FIG. 12 from 1%, 0.1% or 0.01% (Mol/Mol) of mutant in wildtype background after amplification reaction.

TABLE 5 Primers for enrichment of mutant nucleic acid Pool Forward Forward Reverse Reverse ID Primer ID Primer Sequence Primer ID Primer Sequence PMS SMDM13 CAGGAAACAGCTATGAC SMDM13M TGTAAAACGACGGCC 001 CF0033 CGTGGAGAAGCTCCCAA R0033 AGTCGAACGCACCGG CCAAGC AGCT SMDM13 TGTAAAACGACGGCCAG SMDM13C CAGGAAACAGCTATG MF0055 TGAAAGTTAAAATTCCC R0055 ACCGGCCTGAGGTTC GTCGCTATCAAA AGAGCCATG SMDM13 CAGGAAACAGCTATGAC SMDM13M TGTAAAACGACGGCC CF0014 CGAAGCCACACTGACGT R0014 AGTGGCACGTGGGGG GCCTCT TTGTCCACGA SMDM13 CAGGAAACAGCTATGAC SMDM13M TGTAAAACGACGGCC CF0041 CCAGCCAGGAACGTACT R0041 AGTGCACCCAGCAGT GGTGAA TTGGCCC PMS SMDM13 CAGGAAACAGCTATGAC SMDM13M TGTAAAACGACGGCC 002 CF0002 CGTGGAGAAGCTCCCAA R0002 AGTTGCCGAACGCAC CCAAGC CGGAGCA SMDM13 TGTAAAACGACGGCCAG SMDM13C CAGGAAACAGCTATG MF0010 TGAAAGTTAAAATTCCC R0010 ACCGGCCTGAGGTTC GTCGCTATCAAGA AGAGCCATG SMDM13 TGTAAAACGACGGCCAG SMDM13C CAGGAAACAGCTATG MF0044 TCACCGTGCAGCTCATC R0044 ACCGTTGAGCAGGTA AT CTGGGAGCCA SMDM13 CAGGAAACAGCTATGAC SMDM13M TGTAAAACGACGGCC CF0009 CCAGCCAGGIUCGTACT R0009 AGTCTTTCTCTTCCGC GGTGAA ACCCAGCT

Example 3. Enrichment of Mutant Nucleic Acid by Mismatched PAP Primers for Sequencing

In 20 μL PCR reaction solution, a pair of primers (one primer is SMDCR0166 and the other primer is selected from one of SMDMF0166, SMDMF0166G3, SMDMF0166G6, SMDMF0166C9, SMDMF0166C12, SMDMF0166G15) (see Table 6) with each primer containing dideoxynucleotide at its 3′ end in 0.5 μM concentration were added with 2 μL of 10X PCR buffer, with final concentration of 3 mM MgCl₂, 0.2 mM dNTP, 90 μM of pyrophosphate and nits of KlenTaq-S. 30 ng of 100% wild type human genomic DNA (see Table 6) or wild type human genomic DNA spiked with 0.1% mutant genomic DNA (EGFR T790M, see Table 6) was also added to the PCR reaction mixture. The PCR tube was loaded on a thermal cycler and run the following temperature profile: 95° C. for 2 min; 95° C. 15 seconds, 65° C. 120 seconds, for 40 cycles; held at 4° C. 5 μL ExoSAP-IT™ solution (Affymetrix, CA) was added to the tube, and the reaction was incubated at 37° C. for 15 min, 80° C. for 10 min, held at 4° C. 2 μL of treated reaction solution was used to perform cycle sequencing reaction with BigDye™ Terminator v3.1 cycle sequencing kit (Life Technologies, CA) and purified according to manufacturer protocol. The purified sample electrophoresis was carried out on ABI Prism 3730 DNA analyzer according to manufacturer recommended protocol. The results are shown in FIG. 13. It can be seen that the mismatched nucleotide contributes to decreasing false positive results.

TABLE 6 Primer and template sequence for EGFR T790M detection Primer or Template Sequence SMDMF0166 CTCCACCGTGCAGCTCATCAddT SMDMF0166G3 CTCCACCGTGCAGGTCATGAddT SMDMF0166G6 CTCCACCGTGCAGCTGATCAddT SMDMF0166C9 CTCCACCGTGCACCTCATCAddT SMDMF0166C12 CTCCACCGTCCAGCTCATCAddT SMDMF0166G15 CFCCACGGTGCAGCTCATCAddT SMDCR0166 GTTGAGCAGGTACTGGGAGCCddA WT Template GAGGTGGCACGTCGAGTAGTGCGTCGAGTACG (3′ to 5′) GGAAGCCGACGGAGGACCTGATACAGGC--- Mut Template GAGGTGGCACGTCGAGTAGTACGTCGAGTACG (3′ to 5′) GGAAGCCGACGGAGGACCTGATACAGGC---

Example 4. Enrichment of Mutant Nucleic Acid by PAP Primers and Proof-Reading PFU Enzyme for Sequencing

In 20 μL PCR reaction solution, forward and reverse primer pairs (see Table 7) with each primer containing dideoxynucleotide at its 3′ end in 0.5 μM concentration were added with 2 μL of 10X PCR buffer, with final concentration of 3 mM MgCl₂, 0.2 mM dNTP, 90 μM of pyrophosphate and 2 units of KlenTaq-S with or without 2 units of Pfu DNA polymerase (Promega, Wis.) were added to the reaction mixture. Then 30 ng of 100% wild type human genomic DNA (NA12878) (see Table 7) or wild type human genomic DNA (NA12878) spiked with 0.1% mutant genomic DNA (EGFR G719S, see Table 7) was also added to the PCR reaction mixture. The PCR tube was loaded on a thermal cycler and run the following temperature profile: 95° C. for 2 min; 95° C. 15 seconds, 65° C. 120 seconds, for 40 cycles; held at 4° C. 5 μL ExoSAP-IT™ solution (Affymetrix, CA) was added to the tube, and the reaction was incubated at 37° C. for 15 min, 80° C. for 10 min, held at 4° C. 2 μL of treated reaction solution was used to perform cycle sequencing with BigDye™ Terminator v3.1 cycle sequencing kit (Life Technologies, CA) and purified according to manufacturer protocol. The purified sample electrophoresis was carded out on ABI Prism 3730 DNA analyzer according to manufacturer recommended protocol. The results are shown in FIG. 14. It can be seen that the proof-reading PELT enzyme contributes to decreasing false positive results.

TABLE 7 Primer and template sequence for EGFR G719S detection Primer or Template Sequence SMDMF0166 CTCCACCGTGCAGCTCATCAddT SMDMF0166G3 CTCCACCGTGCAGCTCATGAddT SMDMF0166G6 CTCCACCGTGCAGCTGATCAddT SMDMF0166C9 CTCCACCGTGCACCTCATCAddT SMDMF0166C12 CTCCACCGTCCAGCTCATCAddT SMDMF0166G15 CTCCACGGTGCAGCTCATCAddT SMDCR0166 GTTGAGCAGGTACTGGGAGCCddA WT Template GAGGTGGCACGTCGAGTAGTGCGTCGAGTACG (3′ to 5′) GGAAGCCGACGGAGGACCTGATACAGGC--- Mut Template GAGGTGGCACGTCGAGTAGTACGTCGAGTACG (3′ to 5′) GGAAGCCGACGGAGGACCTGATACAGGC--- 

1. A method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid.
 2. The method of claim 1, wherein the blocking group is at or near 3′ terminal of each blocking primer.
 3. The method of claim 1, wherein the blocking group is 2′, 3′-dideoxynucleotide, ribonucleotide residue, 2′, 3′ SH nucleotide, or 2′-O—PO3 nucleotide.
 4. The method of claim 1, wherein the blocking primer is further modified to decrease the amplification of undesired nucleic acid.
 5. The method of claim 4, wherein the modification is introduction of at least one mismatched nucleotide in the primer.
 6. (canceled)
 7. The method of claim 5, wherein the mismatched nucleotide is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 bp away from the nucleotide with the blocking group.
 8. The method of claim 5, wherein the mismatched nucleotide base is located on the 5′ side of the nucleotide with the blocking group.
 9. (canceled)
 10. The method of claim 4, wherein the modification is a modification to form an extra bridge connecting the 2′ oxygen and 4′ carbon of at least one nucleotide of the blocking primer. 11-12. (canceled)
 13. The method of claim 1, wherein the reaction mixture comprises at least 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 different types of primer pairs.
 14. (canceled)
 15. The method of claim 1, wherein the different types of primers pairs can complementarily bind to different target nucleic acids or different sequences in the same target nucleic acid. 16-17. (canceled)
 18. The method of claim 1, wherein the target nucleic acid is double strand DNA ligated with single or double adaptor tags or single stranded DNA ligated with single adaptor tag.
 19. (canceled)
 20. The method of claim 1, wherein the target nucleic acid is double stranded DNA comprising single or double molecular index tag or single stranded DNA comprising single molecular index tag.
 21. The method of claim 20, wherein the molecular index tag comprises unique identifier nucleic acid sequence and an adaptor tag.
 22. (canceled)
 23. The method of claim 1, wherein the primers have common tailing sequence at or near 5′ terminal of the primers.
 24. The method of claim 23, wherein the common tailing sequence can be used as molecular index tag, sample index tag or adaptor tag or combinations of three tags. 25-31. (canceled)
 32. The method of claim 1, wherein the nucleic acid other than the target nucleic acid is not amplified in step (b) substantially.
 33. (canceled)
 34. The method of claim 1, wherein the method is used for selective enrichment of mutant nucleic acid in a sample comprising wildtype nucleic acid.
 35. The method of claim 34, wherein at least one blocking primer is complementary to the mutant nucleic acid at the mutant residues and the nucleotide of the blocking primer corresponding to a mutant residue has the blocking group. 36-39. (canceled)
 40. A method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pairs is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid; (c) adding adaptor tag, molecular index tag and/or sample index tag to the reaction products obtained from step (b); and (d) determining the sequence of the reaction products obtained from step (c). 41-46. (canceled)
 47. A method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least one type of primers that is complementary to a portion of the target nucleic acid, and each type of primers have at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to decrease the amplification of undesired nucleic acid, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primer; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid. 48-57. (canceled) 